PROJECT SUMMARY. Encephalitic arthropod-borne viruses are emerging as a significant cause of death and disability around the world. Understanding the mechanisms by which these viruses induce morbidity and mortality is important so that therapeutic interventions can be developed. Sindbis virus (SINV) is the prototypical alphavirus. While causing mild disease in humans, SINV results in encephalomyelitis in mice, paralleling the disease induced by closely related eastern, western, and Venezuelan equine encephalitis viruses, which cause outbreaks of encephalitis in the Americas. Virulence is dependent on both age and strain of mice as well as the SINV strain used for infection. TE, a SINV laboratory strain, results in fatal encephalomyelitis in suckling C57BL/6 (B6) mice and nonfatal encephalomyelitis in adult mice, making TE useful for studying clearance of viral RNA from the central nervous system (CNS). Our lab performs both intracranial (IC) and intranasal (IN) inoculations to infect mice with SINV. Preliminary studies show that while all B6 mice recover from TE infection after IC inoculation, 20-30% die following IN inoculation. Almost all of these moribund mice have gastric ulceration and intestinal bleeding, however SINV RNA is only detected within CNS tissue. Cytokine levels in the CNS and serum do not differ between moribund and sick mice. The primary goal of this proposal is to determine the differences in viral distribution and pathogenesis between mice that die and mice that recover following IN inoculation with TE. First, the distribution of viral antigen and RNA following IN and IC inoculation with SINV TE in B6 mice will be determined for 2 weeks post infection. Differences in distribution of viral antigen and RNA will be compared between moribund and sick mice following IN inoculation to determine if specific locations are infected when mice become moribund. Because TE infects neurons within the brainstem at these time points, the brainstem will be a focus. Second, specific brainstem pathology will be compared to determine if there is a correlation with severe disease resulting from autonomic dysfunction in moribund mice. Differences in immune-mediated and viral-induced pathological changes within the brainstem will be compared between moribund and sick mice. Potential autonomic dysfunction as a cause of death will then be assessed in infected mice by measuring heart rate and blood pressure variability as direct measures of autonomic nervous system function. Gastrin levels in TE-infected mice will be measured to determine if levels are increased and lead to excessive acid secretion and gastric ulceration further indicating brainstem dysfunction in moribund mice. Lastly, viral antigen and RNA within the brainstem will be analyzed to determine if disruption of specific structures leads to autonomic dysfunction and correlates with TE-induced mortality. Determining the mechanism by which a nonfatal SINV strain only results in mortality following IN inoculation will increase our understanding of the mechanisms of viral propagation within the CNS that lead to mortality and identify relevant therapeutic interventions.